This project focuses on the role of the NS3/4A protease of HCV in disrupting innate cellular antiviral defenses by mediating proteolysis of novel cellular substrates that function as signaling proteins. Our overarching goals include a better understanding of how sequence variation within NS3/4A influences this and how disruption of signaling contributes to cellular permissiveness for HCV replication. The proposed studies build on a long-term and productive effort to define critical HCV-host cell interactions that contribute to the pathogenesis of hepatitis C. During the current funding period, we have developed cell culture systems supporting robust replication of HCV RNA and demonstrated that RNA replication suppresses signaling pathways that are normally activated by viruses. In collaboration with the Gale laboratory (Project 4), we have shown that the NS3/4A protease disrupts two distinct virus-activated cellular signaling pathways, one initiated by dsRNA engagement of Toll-like receptor 3 (TLR3) and the other by recognition of structured viral RNA by the cellular DExH RNA helicase, retinoic acid-inducible gene I (RIG-I). Both pathways lead to activation of interferon regulatory factor 3 (IRF-3) and NF-kappaB, and thus act to induce expression of type 1 interferon, numerous interferon-stimulated genes, proinflammatory cytokines and chemokines which collectively exert a cellular antiviral effect. Our central hypothesis is that disruption of these pathways by NS3/4A contributes significantly to cellular permissiveness for HCV as well as persistence of infection in humans and is thus a target worthy of therapeutic intervention. Our aims include characterization of the proteolysis of cellular signaling proteins by NS3/4A and the impact of amino acid sequence variation within NS3/4A on disruption of IRF-3 signaling pathways. We will also study the impact of cell culture-adaptive mutations on HCV replication in hepatocytes in vivo, and leverage our growing understanding of the disruption of signaling pathways by HCV with recent advances in RNA replication in vitro to develop cell culture systems that are fully permissive for the virus.